A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase (SELD). A cysteine residue (Cys-17) that is essential for catalytic activity of the enzyme is located in a glycine-rich segment near the N-terminus of the protein. The possibility that this peptide sequence (HGAGCGCK) defines the ATP- binding site of the enzyme, as does a conserved ATP or GTP binding sequence (GXXXXGKS/T) found in several other proteins, was tested by site-specific mutagenesis. Thus, His-13 and Gly-18 were changed to Asn and Val, respectively, and Lys-20 to Arg or Gln. Catalytic activity was markedly decreased by mutation of Lys-20 to Arg and abolished by mutation of Lys-20 to Gln. The mutation of Cys-19 and His-13 did not substantially alter the ATP Km and Vmax values whereas the Gly-18 mutation resulted in a four-fold increase in the ATP Km value compared to that of the wild type. ATP-binding properties of the mutant enzymes were determined using Mn-[32P]ATP or Mn-[14C]ATP and gel filtration. Photo-affinity labeling of the proteins with [gamma-32P]8-azido-ATP showed that all mutant enzymes could be labeled with the ATP-analog except those in which Cys-17 or Cys-19 were replaced with serine.